Hematopoietic stimulation

ABSTRACT

Methods and products for stimulating hematopoiesis, preventing low levels of hematopoietic cells and producing increased numbers of hematopoietic and mature blood cells are provided. The methods and products can be used both in vivo and in vitro. The methods involve administering an agent of Formula I:                    
     wherein m is an integer between 0 and 10, inclusive; A and A 1  are L-amino acid residues such that the A in each repeating bracketed unit can be the same or a different amino acid residue; the C bonded to B is in the L-configuration; the bonds between A and N, A 1  and C, and between A 1  and N are peptide bonds; and each X 1  and X 2  is, independently, a hydroxyl group or a group capable of being hydrolyzed to a hydroxyl group in aqueous solution at physiological pH. A particularly preferred agent that is useful in practicing the invention is a ValBoroPro.

RELATED APPLICATIONS

This application is a continuation application of prior U.S. patentapplication Ser. No. 09/304,199, filed May 3, 1999, entitledHematopoietic Stimulation, now issued as U.S. Pat. No. 6,300,314 B1 theentire contents of which are incorporated herein by reference and whichclaims priority under 35 U.S.C. § 119(e) to U.S. provisional applicationserial No. 60/084,128 filed May 4, 1998.

BACKGROUND OF THE INVENTION

The present invention relates to methods and products for producingincreased numbers of hematopoietic cells, of restoring to preselectednormal levels numbers of hematopoietic cells, to therapies for treatingdeficiencies in hematopoietic cells and to in vitro methodologies forculturing hematopoietic cells.

PT-100 is a dipeptide consisting of valine-prolineboronic acid(ValboroPro) designed to interact with the cell surface receptor CD26.CD26, a type II transmembrane protein is expressed on the cell surfaceof a number of cell types, including lymphocytes (Marguet, D. et al.,Advances in Neuroimmunol. 3:209-215 (1993)), hematopoietic cells(Vivier, I. et al., J. Immunol. 147:447-454 (1991); Bristol, et al., J.Immunol. 149:367 (1992)) thymocytes (Dang, N. H. et al., J. Immunol.147:2825-2832 (1991), Tanaka, T. et al., J. Immunol. 149:481-486 (1992),Darmoul, D. et al., J Biol. Chem. 267:4824-4833 (1992)), intestinalbrush border membrane and endothelial cells. Cell surface associatedCD26 is a sialoglycoprotein, with most of its mass on the outside of thecell.

CD26 has been best characterized on peripheral T cells where itfunctions as a potent costimulatory signal for T cell activation. Itssurface expression is upregulated upon T cell activation (Dong, R. P. etal., Cell 9:153-162 (1996), Torimoto, Y. et al., J. Immunol 147:2514(1991), Mittrucker, H-W. et al., Eur. J Immun. 25:295-297 (1995),Hafler, D. A. et al, J. Immunol. 142:2590-2596 (1989), Dang, N. H. etal., J. Immunol 144:409 (1990)). CD26 has also been identified inrodents as an important regulatory surface receptor in hematopoiesis andlymphoid development (Vivier, I. et al., J. Immunol. 147:447-454(1991)). The primary structure of CD26 is highly conserved betweenspecies (Ogata, S. et al., J. Biol. Chem. 264:3596-3601 (1998)). Inhumans CD26 seems to be involved in the regulation of thymocyteactivation, differentiation and maturation (Dang, N. H. et al., J.Immunol. 147:2825-2832 (1991); Kameoka, J. et al., Blood 85:1132-1137(1995)). We have evidence that CD26 is expressed within the human andmurine hematopoietic systems.

CD26 is an ectoenzyme with activity identical to that of DipeptidylPeptidase IV (DPP-IV), a serine type exopeptidase with high substratespecificity. It cleaves N-terminal dipeptides from proteins if thepenultimate amino acid is proline, or in some cases alaninie (Fleischer,B. Immunol. Today 15:180 (1994)). PT-100 is a potent inhibitor of DPP-IVactivity.

The prior art PCT published application WO94/03055 teaches methods ofproducing increased numbers of hematopoietic cells by administeringinhibitors of DPP-IV. The teaching of this published application,however, is that dosages of at least 1 mg/kg body weight are necessaryto achieve such hematopoietic cell increases. This published applicationalso teaches that inhibitors are administered to mammals which have anestablished deficiency of hematopoietic cells. The teaching alsosuggests that cytokines be administered in conjunction with theinhibitors to increase the production of hematopoietic cells in asubject.

SUMMARY OF THE INVENTION

The invention is based upon a variety of surprising and unexpectedfindings. It has been discovered, unexpectedly, that the agents usefulaccording to the invention stimulate growth factor production by stromalcells. It also has been discovered, unexpectedly, that the agents usefulaccording to the invention stimulate proliferation of primitivehematopoietic progenitor cells, but do not stimulate directly thedifferentiation or proliferation of committed progenitor cells. Itfurther has been discovered, unexpectedly, that the agents usefulaccording to the invention can be administered at doses much lower thanwould have been expected according to the teachings of the prior art.Another unexpected finding is that the agents according to the inventioncan accelerate the time it takes to achieve hematopoietic cell recoveryafter treatment with an hematopoietic cell inhibitor. Another unexpectedfinding is that the agents useful according to the invention can atrelatively low doses, restore normal levels of neutrophils at least asfast as the most successful commercially available product usedworldwide for this purpose, except that the agents useful according tothe invention can be used orally, whereas the commercially availableproduct (which represents more than a billion dollar market) must beinjected. These unexpected results have important therapeutic andexperimental research implications.

According to one aspect of the invention, a method is provided fortreating a subject to stimulate hematopoiesis in the subject. Theinvention involves administering to a subject in need of such treatmentan amount of an agent effective to increase the number of hematopoieticcells or mature blood cells in the subject, wherein the amount is lessthan 1 mg/kg body weight per day and wherein the agent is a compound ofFormula I.

The agents useful according to the invention are compounds of Formula I:

wherein m is an integer between 0 and 10, inclusive; A and A₁ areL-amino acid residues (for glycine there is no such distinction) suchthat the A in each repeating bracketed unit can be a different aminoacid residue; the C bonded to B is in the L-configuration; the bondsbetween A and N, A₁ and C, and between A₁ and N are peptide bonds; andeach X, and X₂ is, independently, a hydroxyl group or a group capable ofbeing hydrolysed to a hydroxyl group in aqueous solution atphysiological pH. By “the C bonded to B is in the L-configuration” ismeant that the absolute configuration of the C is like that of anL-amino acid.

Thus, the

group has the same relationship to the C as the —COOH group of anL-amino acid has to its α carbon. In some embodiments, A and A₁ areindependently proline or alanine residues; m is 0; X₁ and X₂ arehydroxyl groups; the inhibitor is L-Ala-L-boroPro; and the inhibitor isL-Pro-L-boroPro.

In one important aspect of the invention, the subject has an abnormallylow level of hematopoietic cells or mature blood cells and the agent isadministered in an amount effective to restore levels of a hematopoieticcell-type or mature blood cell-type to a preselected normal orprotective level. The agent preferably is administered to the subject inat least 2 doses in an 18 hours period. The invention has particularlyimportant applications in the restoration of normal or protective levelsof neutrophils, erythrocytes and platelets. The most preferred agent isValBoroPro.

According to another aspect of the invention, a method is provided forshortening or eliminating the time that a subject has an abnormally lowlevel of hematopoietic or mature blood cells resulting from treatmentwith a hematopoietic cell inhibitor. An agent is administered to asubject in need of such treatment in an amount effective to increase thenumber of hematopoietic cells or mature blood cells in the subject,wherein the administration of the agent begins prior to or substantiallysimultaneous with administration of the hematopoietic cell inhibitor.The agents and the preferred agent are as described above. In oneimportant embodiment, the hematopoietic cell inhibitor causes anabnormally low level of hematopoietic cells or mature blood cells in thesubject and the agent is administered in an amount effective to restorelevels of a hematopoietic cell type to a preselected normal orprotective level. Preferably, the agent is administered to the subjectin at least 2 doses in an 18 hour period. In important embodiments, theagent is used to restore in the subject normal or protective levels ofneutrophils, erythrocytes or platelets. The preferred effective amountof agent is as described above.

According to another aspect of the invention, a method is provided forpreparing a subject for treatment with a hematopoietic cell inhibitor.The method involves administering to the subject prior to the subjectreceiving the hematopoietic cell inhibitor an agent in an amounteffective to stimulate in the subject production of growth factors. Inone embodiment the agent stimulates stromal cell production of growthfactor. The agents and the preferred agent are as described above. Inone important embodiment, the growth factor is granulocyte colonystimulating factor. In other embodiments the growth factor is selectedfrom the group consisting of IL-1, IL-2, IL-3, IL-4, IL-6, IL-11, IL-17,TPO, EPO, MCSF, GMCSF, FLT-3 Ligand and Stem Cell Factor. Preferably,the amount administered to the subject is less than 1 mg/kg body weightper day. It also is preferred that the administration of the agent be inat least 2 doses of the agent in an 18 hour period.

According to another aspect of the invention, a method is provided fortreating a subject to increase the number of hematopoietic cells ormature blood cells in the subject. An agent is administered to a subjectin need of such treatment in an amount effective to increasehematopoietic cells or mature blood cells in the subject, wherein theagent is administered in a first regimen consisting of 2 doses or 3doses in an 18 hour period. The agents and the preferred agent are asdescribed above. In one important embodiment, the agent is administeredin a second regimen consisting of 2 doses or 3 doses in an 18 hoursperiod, wherein the second regimen is separate in time from the firstregimen. In another embodiment, the agent is administered in a thirdregimen consisting of 2 doses or 3 doses in an 18 hour period, whereinthe third regimen is separate in time from the first and secondregimens. In other embodiments, the agent is administered optionally ina fourth regimen, a fifth regimen, a sixth regimen, or a seventhregimen, wherein each of such regimens consists of 2 doses or 3 doses inan 18 hours period, and wherein the regimens are separate in time fromone another and from the prior regimens. In one important embodiment,the subject has an abnormally low neutrophil count and the amount iseffective to restore in the subject a preselected level of neutrophils.In other important embodiments the subject has abnormally low levels oferythrocytes and platelets. The preferred dosages, agents, and the likeare as described above. In important embodiments, the dosage is no morethan six regimens, no more than five regimens, no more than fourregimens, no more than three regimens, and even no more than tworegimens.

According to another aspect of the invention, a method is provided forpreparing a subject's cells for reintroduction into the subject. Themethod involves treating the subject with an agent in an amounteffective to stimulate in the subject the hematopoietic cells, thencollecting the hematopoietic cells from the subject. The collected cellslater are reintroduced into the subject. The collected cells optionallycan be ex vivo cultured. The agents and preferred agent are as describedabove. In one embodiment, the ex vivo culturing is carried out in thepresence of an amount of the agent effective to stimulate proliferationof the collected cells. In another embodiment, the concentration of theagent in medium surrounding the collected cells is less than 10⁻⁸ molesper liter, and less than 10⁻⁹ moles per liter and even less than 10⁻¹⁰moles per liter.

According to another aspect of the invention, a method is provided forstimulating growth factor production by stromal cells. The methodinvolves contacting the stromal cells with an agent in an amounteffective to stimulate growth factor production by the stromal cells.The agents and the preferred agent are as described above. In oneembodiment, the stromal cells are in an in vitro layer of stromal cellsfor supporting early progenitor cell growth and further comprisingculturing the stem cells in the presence of these stromal cells. Inanother embodiment, the stromal cells are in vivo in a subject. Inanother embodiment, the growth factor is granulocyte colony stimulatingfactor. In other embodiments the growth factor is selected from thegroup consisting of IL-1, IL-2, IL-3, IL-4, IL-6, IL-11, IL-17, TPO,EPO, MCSF, GMCSF, FLT-3 Ligand and Stem Cell Factor. In an in vivoembodiment, the agent is administered to a subject in an amount lessthan 1 mg/kg body weight per day. In still another embodiment, the stemcells are cultured in an environment free of exogenously addedgranulocyte colony stimulating factor. In important embodiments thestromal cells are bone marrow or thymic stromal cells.

According to another aspect of the invention, a kit is provided fortreating a subject having an abnormally low level of hematopoietic cellsresulting from treatment with a hematopoietic cell inhibitor or fortreating prophylactically a subject being treated with a hematopoieticcell inhibitor to prevent decrease or loss of hematopoietic and/ormature blood cells. The kit is a package containing a first dosage andinstructions for treating a subject substantially simultaneous with orprior to treatment with the hematopoietic cell inhibitor. The packagealso contains a second dosage and instructions for treating a subjectonly after treatment with the hematopoietic cell inhibitor. The dosagesare in effective amounts and the agents and preferred agent are asdescribed above. In one embodiment, the second dosage is between 2 and 5regimens, each of the regimens consisting of 2 or 3 doses per day of theagent. In one embodiment, the combination of the doses is less than 1mg/kg body weight per day. One preferred kit is for treatment ofneutropenia. Other preferred kits are for treatment of an abnormally lowlevel of erythrocytes or platelets.

According to still another aspect of the invention, a kit is providedfor treating a subject having abnormally low level of hematopoieticcells. The kit is a package containing a complete dosage for restoringnormal levels of a hematopoietic cell type. The package consistsessentially of: (1) a first dosage in an effective amount foradministration to the subject during a first day, (2) a second dosage inan effective amount for administration to the subject during a secondday, (3) optionally, a third dosage in an effective amount foradministration to the subject during a third day, (4) optionally, afourth dosage in an effective a mount for administration to the subjectduring a fourth day, (5) optionally, a fifth dosage in an effectiveamount for administration to the subject during a fifth day, (6)optionally, a sixth dosage in an effective amount for administration tothe subject during a sixth day and (7) optionally, a seventh dosage inan effective amount for administration to the subject during a seventhday. The agents and preferred agent are as described above. In oneimportant embodiment, each of the dosages consists of 2 or 3 doses ofthe agent for administration each day. Preferred doses and dosages areas described above. In important embodiments, the kit consistsessentially of less than 5, less than 4, and less than 3 and even lessthan 2 dosages.

These and other aspects of the invention will be described in greaterdetail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a medicinal package for administering a 5 day medicinal courseof treatment for treating myelosuppression or anemia resulting fromcancer chemotherapy.

FIG. 2 is a graph depicting the regeneration of neutrophils incyclophosphamide-treated mice. PT-100 at indicated doses or salineadministered by gavage. Absolute neutrophil counts in mice not treatedwith cyclophosphamide are on the average 190×10⁴ cells/ml as indicatedby the dashed horizontal line.

FIG. 3 is a graph depicting the regeneration of neutrophils incyclophosphamide-treated mice in response to subcutaneous administrationof PT-100. Saline or PT-100 were administered b.i.d. for 5 consecutivedays. The average absolute neutrophil count in mice not treated withcyclophosphamide was 185×10⁴ cells/ml as indicated by the horizontaldashed line.

FIG. 4 is a graph depicting the regeneration of neutrophils incyclophosphamide-treated mice in response to PT-100 and granulocytecolony stimulating factor. PT-100 in saline was administered by gavage,and GCSF by subcutaneous injections, for 5 days. Absolute neutrophilcount in mice not treated with cyclophosphamide are on average 190×10⁴cells/ml as indicated by the dashed horizontal line.

FIG. 5 is a graph depicting the effect of PT-100 dose number on theregeneration of neutrophils in cyclophosphamide-treated mice. PT-100, atindicated concentrations, was administered either once or twice per daysubcutaneously for 5 days. The average absolute neutrophil count formice not treated with cyclophosphamide was 200×10 ⁴ cells/ml asindicated by the dashed horizontal line.

FIG. 6 is a graph depicting the effect of duration of PT-100administration on absolute neutrophil count and rate of neutrophilrecovery in cyclophosphamide-treated mice. PT-100 (5 μg/b.i.d.) wasadministered to cyclophosphamide-treated mice by gavage for theindicated length of time. The dashed horizontal line indicates theaverage absolute neutrophil count for mice not treated withcyclophosphamide.

FIG. 7 is a graph showing the effect of duration of PT-100 treatment onthe regeneration of neutrophils in cyclophosphamide-treated mice. PT-100(2 μg/b.i.d.), or saline were administered by gavage for the indicatedduration. The average absolute neutrophil count for mice not treatedwith cyclophosphamide was 194×10⁴ cells/ml as shown by the dotted line.

FIG. 8 is a graph depicting the colony formation ability of cells inresponse to PT-100 in a long-term culture (LTC) assay. Human bone marrowcells were incubated in LTC for 4 weeks in the absence or presence ofindicated amounts of PT-100, followed by a 2 week culture in semi-solidmedium.

FIG. 9 is a graph showing that PT-100 stimulates hematopoiesis in thespleen of normal mice.

FIG. 10 is a graph showing that PT-100 stimulates production of G-CSF byhuman stromal cells.

DETAILED DESCRIPTION OF THE INVENTION

The invention involves the stimulation of proliferation, differentiationand mobilization of hematopoietic cells. The invention is usefulwhenever it is desirable to stimulate the proliferation ordifferentiation, of or to mobilize, hematopoietic cells. Mobilization ofhematopoietic cells is characterized by the enrichment of earlyprogenitor cells in the bone marrow and the recruitment of these cellsto the periphery in response to a mobilization agent (e.g. G-CSF,GM-CSF, etc.). The agents useful according to the invention can be usedto inhibit hematopoietic cell deficiencies or to restore hematopoieticand mature blood cell count in subjects with such deficiencies. Suchagents also may be used in connection with hematopoietic celltransplants, such as bone marrow or peripheral blood transplants, whenused to replenish or create an immune system in a subject. The agentsfurther can be used as an immune booster. The agents also are useful invitro in connection with the culturing of cells for therapeutic andresearch uses.

As used herein, subject means humans, nonhuman primates, dogs, cats,sheep, goats, horses, cows, pigs and rodents.

One important aspect of the invention involves restoring or preventing adeficiency in hematopoietic cell number in a subject. Such deficienciescan arise, for example, from genetic abnormalities, from disease, fromstress, from chemotherapy (e.g. cytotoxic drug treatment, steroid drugtreatment, immunosuppressive drug treatment, etc.) and from radiationtreatment.

The invention is useful in general to restore deficiencies created byhematopoietic cell inhibitors. A hematopoietic cell inhibitor is anexogenously-applied agent (such as a drug or radiation treatment) whichcauses a decrease in the subject of hematopoietic cells and/or matureblood cells.

Hematopoietic cells as used herein refer to granulocytes (e.g.promyelocytes, neutrophils, eosinophils and basophils), erythrocytes,reticulocytes, thrombocytes (e.g. megakaryoblasts, platelet-producingmegakaryocytes and platelets), lymphocytes, monocytes, dendritic cellsand macrophages. Mature blood cells consist of mature lymphocytes,platelets, erythrocytes, reticulocytes, granulocytes and macrophages. Incertain important aspects of the invention, the agents useful accordingto the invention increase the number of neutrophils, erythrocytes andplatelets. In connection with neutrophils, the agents may be used totreat, inter alia, drug or radiation-induced neutropenia, chronicidiopathic neutropenia and cyclic neutropenia.

One important aspect of the invention is restoring in a subject “normal”or “protective” hematopoietic cell levels. A “normal” level as usedherein can be a level in a control population, which preferably includessubjects having similar characteristics as the treated individual, suchas age. The “normal” level can also be a range, for example, where apopulation is used to obtain a baseline range for a particular groupinto which the subject falls. The population can also be divided intogroups, such as into quadrants, with the lowest quadrant beingindividuals with the lowest levels of hematopoietic cells and thehighest quadrant being individuals having the highest levels ofhematopoietic cells. Thus, the “normal” value can depend upon aparticular population selected. Preferably, the normal levels are thoseof apparently healthy subjects which have no prior history ofhematopoietic cell disorders. Such “normal” levels, then can beestablished as preselected values, taking into account the category inwhich an individual falls. Appropriate ranges and categories can beselected with no more than routine experimentation by those of ordinaryskill in the art. Either the mean or another preselected number withinthe range can be established as the normal preselected value. Likewise,the level in a subject prior to treatment with a hematopoietic cellinhibitor can be used as the predetermined value.

In general, the normal range for neutrophils is about 1800-7250 per μl(mean −3650); for basophils 0-150 per μl (mean −30); for eosinophils0-700 per μl (mean −150); for macrophages and monocytes 200-950 per μl(mean −430); for lymphocytes 1500-4000 per μl (mean −2500); forerythrocytes 4.2×10⁶−6.1×10⁶ per μl; and for platelets 133×10³−333×10³per μl. The foregoing ranges are at the 95% confidence level.

In connection with certain conditions, the medical community hasestablished certain preselected values. For example, mild neutropenia ischaracterized as having a count of between 1000 and 2000 per μl,moderate neutropenia at between 500 and 1000 per μl and severeneutropenia at below 500 per μl. Likewise, in adults, a lymphocyte countat less than 1500 is considered a medically undesirable condition. Inchildren the value is less than 3000. Other preselected values will bereadily known to those of ordinary skill in the art. The agents usefulaccording to the invention can be used to establish or to re-establishsuch preselected values, including normal levels.

Protective levels of hematopoietic cells is the number of cells requiredto confer clinical benefit to the patient. The required levels can beequal to or less than “normal levels”. Such levels are well known tothose of ordinary skill in the art. For example, a protective level ofneutrophils is above 1000, preferably, at least 1500.

According to another aspect of the invention, the agents useful hereincan be applied at doses below those which were described in the priorart. In particular, it has been discovered unexpectedly that the agentsof the invention can be administered in doses less than 1 mg/kg bodyweight per day. In particular, the agents of the invention have beenused successfully at levels of 0.1 mg/kg body weight per day, which isone order of magnitude below the teachings of the prior art. As will bereadily recognized by those of ordinary skill in the art, this hasadvantages in that less material is required for treatment, therebylessening any risk of side effects. Likewise, this has advantages inconnection with the cost of manufacture of the drug products of theinvention.

According to another aspect of the invention, better therapeutic resultscan be achieved when the agents are applied in multiple doses per day.This finding is unexpected and, additionally, it has been found thatthere is no added medically useful effect when the agents usefulaccording to the invention are administered for lengthy periods of time.Thus, it has been discovered, unexpectedly, that only very brief periodsof treatment are needed to achieve established therapeutic goals.

As described in the examples below, subjects treated with the agentsuseful according to the invention in 2 doses per day versus 1 dose perday achieved recovery of hematopoietic cells almost 33% faster thansubjects receiving only 1 dose per day. Surprisingly, this result didnot depend upon the absolute amount of drug given to the subject, butinstead related to the number of times the subject was administered thedrug. In other words, as shown below, giving twice as much drug, butonly once a day, did not speed the recovery of hematopoietic cellnumber. Thus, an aspect of the invention involves giving the agentsuseful according to the invention in 2 or 3 doses in an 18 hour period.As used herein, an 18 hour period refers in general to the time duringwhich a subject is awake in any 24 hour period; it is intended toindicate 2 doses per day, 3 doses per day, and the like.

According to still another aspect of the invention, it has beendiscovered unexpectedly that the agents useful according to theinvention need be administered for fewer days than expected according tothe prior art. In particular, in the mouse models employed, there wasvery little difference in the speed of recovery of hematopoietic cellcount and in the ability to reestablish normal levels of hematopoieticcells when treatment was 3 days, versus 4 days, versus 5 days. It isbelieved, therefore, that when applied to humans, a complete drugtreatment will involve 7 days or less, more preferably 6 days or less,more preferably 5 days or less, more preferably 4 days or less, and evenmore preferably 3 days or less. As a result, the invention thereforeprovides kits which contain complete treatment packages for restoringhematopoietic cell count, which kits are described in greater detailbelow.

According to another aspect of the invention, the time that a subjecthas an abnormally low level of hematopoietic cells resulting fromtreatment with a hematopoietic cell inhibitor is shortened. It has beendiscovered, unexpectedly, that the agents used according to theinvention stimulate growth factor production by stromal cells. Forexample, granulocyte colony stimulating factor (GCSF) production bystromal cells is stimulated. GCSF acts to drive specificallyneutrophil-lineage differentiation. It does not affect thedifferentiation or proliferation of other committed hematopoietic cells,including other granulocytes, such as eosoniphils, basophils, mast cellsand macrophages. (It is known to act synergistically, however, in vitrowith other cytokines to affect proliferation of pluripotent stem cells,though the in vivo importance of this observation is not known). Becausestromal cells are not rapidly dividing cells and are not generallyadversely impacted by hematopoietic cell inhibitors, the agents usefulaccording to the invention can be applied to subjects substantiallysimultaneously with or even prior to treatment with a hematopoietic cellinhibitor in order to stimulate stromal cells to produce growth factorwhich will be readily abundant and helpful in regenerating thehematopoietic cells after treatment by the hematopoietic cell inhibitor.In the prior art, such treatment has been delayed until substantiallyafter treatment with the hematopoietic cell inhibitor. Substantiallysimultaneously with, as used herein, means within 24 hours of treatmentwith the hematopoietic cell inhibitor. Preferably, the agents usefulaccording to the invention are administered within 2 hours of treatmentwith the hematopoietic cell inhibitor, if they are administered aftertreatment with the hematopoietic cell inhibitor. If they areadministered before treatment with the hematopoietic cell inhibitor,then they are administered close enough in time to the treatment withthe inhibitor so that stromal cell production of growth factor isenhanced in the days immediately following treatment with thehematopoietic cell inhibitor.

Another aspect of the invention involves treatment of a subject toprepare a subject for subsequent treatment with other agents. It hasbeen discovered, unexpectedly, that the agents useful according to theinvention stimulate the proliferation of primitive, noncommittedhematopoietic progenitor cells, but not directly the differentiation ofcommitted progenitor cells. It is known in the art that such cells mayor may not include CD34⁺ cells. CD34⁺ cells are immature cells presentin blood products, express the CD34 cell surface marker, and arebelieved to include a subpopulation of cells with the capacity toself-renew and to differentiate into all of the mature blood cell types.Because the agents useful according to the invention stimulate theproliferation of such self-renewing cells, the invention is useful toprepare a subject for treatment with other exogenous growth factors andcytokines which in turn result in the differentiation of suchuncommitted progenitor cells into committed progenitor cells. Likewise,the agents useful according to the invention can be administered to asubject to expand in the subject hematopoietic cells and to mobilizesuch cells, prior to extracting the cells from the subject fortransplantation or re-infusion. Such cells may be used for researchpurposes or can be treated ex vivo or reintroduced into the subject withor without expansion in vitro.

The agents useful according to the invention can be administered inconjunction with exogenous growth factors and cytokines which arespecifically selected to achieve a particular outcome. For example, ifit is desired to stimulate a particular hematopoietic cell type, thengrowth factors and cytokines which stimulate proliferation anddifferentiation of such cell type are used. Thus, it is known thatinterleukins-1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13 and 17 are involvedin lymphocyte differentiation. Interleukins 3 and 4 are involved in mastcell differentiation. Granulocyte macrophage colony stimulating factor(GMCSF), interleukin-3 and interleukin-5 are involved in the eosinophildifferentiation. GMCSF, macrophage colony stimulating factor (MCSF) andIL-3 are involved in macrophage differentiation. GMCSF, GCSF and IL-3are involved in neutrophil differentiation. GMSCF, IL-3, IL-6, IL-11 andTPO are involved in platelet differentiation. Flt3 Ligand is involved indendritic cell growth. GMCSF, IL-3, and erythropoietin are involved inerythrocycte differentiation. Finally, the self-renewal of primitive,pluripotent progenitor cells capable of sustaining hematopoiesisrequires SCF, Flt3 Ligand, G-CSF, IL-3, IL-6 and IL-11. Variouscombinations for achieving a desired result will be apparent to those ofordinary skill in the art. Because the agents useful according to theinvention stimulate primitive, non-committed hematopoietic progenitorcells, they can be used in connection with any of the foregoingcategories of agents to stimulate specifically the proliferation of aparticular hematopoietic cell type. The foregoing factors are well knownto those of ordinary skill in the art, and most are commerciallyavailable.

The invention also lends itself to a variety of in vitro uses.Hematopoietic progenitor cells are preserved or expanded, or theircolony forming unit potential increased, in vitro. One benefit that canbe obtained according to the invention is the stimulation ofhematopoietic progenitor cells by the agents useful according to theinvention. Another benefit that can be obtained is the effect that theagent can have on stromal cells used in in vitro culturing ofhematopoietic progenitor cells. In vitro culturing of hematopoieticcells is often carried out in the presence of stromal cells.Hematopoietic progenitor cells typically will not survive, proliferateor differentiate for very long periods of time in vitro withoutappropriate growth factor support.

Stromal cell layers are used to supply such growth agents to culturedhematopoietic cells, either by culturing the hematopoietic progenitorcells in vitro with such stromal cells or by supplying the hematopoieticprogenitor cells with stromal cell-conditioned medium. The agents usefulaccording to the present invention can be used to treat such stromalcells to cause the stromal cells to manufacture and release growthfactors. The incubation of stromal cells with the agents usefulaccording to the invention and in medium is for a period of timesufficient to allow the stromal cells to secrete factors into themedium. The medium then can be used to supplement the culture ofhematopoietic progenitor cells and other hematopoietic cells.

The culture of hematopoietic cells is with media which is conventionalfor culturing cells. Examples include RPMI, DM, ISCOVES, etc. Theconditions for such culturing also are known to those of ordinary skillin the art. The conditions typically refer to a combination ofparameters (e.g. temperature, CO₂ and O₂ content, nutritive media,etc.). The time sufficient to increase the number of cells is a timethat can be easily determined by a person skilled in the art, and canvary depending on the original number of cells seeded and the amountadded of growth factors and agents useful according to the invention.

The colony forming potential of hematopoietic uncommitted progenitorcells can be increased by in vitro culturing of hematopoietic cells. Thecells can be obtained from any blood product or organ containing cellsof hematopoietic origin. Crude or unfractionated blood products can beenriched for cells having hematopoietic progenitor cell characteristicsin ways well known to those of ordinary skill in the art, prior to orafter culture with the agents useful according to the invention.

A particularly important aspect of the invention is in the use of theagents for treatment of neutropenia. A combination of unexpected resultsmakes the invention particularly useful in the treatment of neutropenia.Firstly, the agents according to the invention can stimulate theproliferation of uncommitted progenitor cells. Secondly, the agentsaccording to the invention also stimulate stromal cells to make GCSF,which is the growth factor critical in the differentiation andproduction of neutrophils per se. Thus, the patient has the dual benefitof stimulation of progenitor cells and differentiation of those cellsinto neutrophils using the agents useful according to the invention.Similar effects are shown with erythrocytes and platelets. Thus,treatment to restore neutrophils, erythrocytes and platelets form anindependent and distinct aspect of the invention, based on theunexpected findings described above.

The invention also involves kits for housing an entire medicinal courseof treatment for a hematopoietic cell deficiency such as neutropenia. Asdiscussed above, it has been discovered surprisingly that the number ofdoses per day and the number of doses overall affect favorably therecovery of hematopoietic cells after treatment with a hematopoieticcell inhibitor. These unexpected findings lend themselves to thedevelopment of a medicinal dispenser which houses an entire medicalcourse of treatment using the agents useful according to the invention.Patient compliance therefore will be enhanced, and an entireprescription can be contained in a single package. Ordinarily, apharmacist individually fills a dispenser unit with a medicament oncethe pharmacist receives a doctor's prescription. Because the dispenserof the invention includes an entire medicinal course of treatment andcan always include a specific number of solid oral dosage forms, thepackage can be pre-filled with the appropriate number of units ofmedicament for treatment for a particular medical purpose.

The medicinal dispenser is a package defining a plurality of medicinalstorage compartments, each compartment for housing an individual unit ofmedicament. An entire medicinal course of treatment is housed in aplurality of medicinal storage compartments.

A package defining a plurality of medicinal storage compartments may beany type of disposable pharmaceutical package or card which holdsmedicaments in individual compartments. Preferably the package is ablister package constructed from a card, which may be made from stiffpaper material, a blister sheet and backing sheet. Such cards are wellknown to those of ordinary skill in the art.

FIG. 1 shows a medicinal dispenser (1) for housing a preferred entiremedicinal course of treatment for neutropenia. The day indicia (2)indicate which day the individual units of medicament are to be taken.These are marked along a first side of the medicinal package. The doseindicia (3) is marked along a second side of the medicinal packageperpendicular to the first side of the medicinal package and indicatesthe time which the individual unit of medicament should be taken. Theunit doses (4) are contained in the dispenser which is a blister pack.This particular package shows a 5 day course of treatment, with 2 dosesper day.

The pharmaceutical preparations, as described above, are administered ineffective amounts. The effective amount will depend upon the mode ofadministration, the particular condition being treated and the desiredoutcome. It will also depend upon, as discussed above, the stage of thecondition, the age and physical condition of the subject, the nature ofconcurrent therapy, if any, and like factors well known to the medicalpractitioner. For therapeutic applications, it is that amount sufficientto achieve a medically desirable result. In some cases this is anyincrease in hematopoietic cell count or mature blood cell count. Inother cases, it will be an increase to a preselected level.

The invention is useful in one aspect to ameliorate the effects oftreatment with a hematopoietic cell inhibitor. If the agents are usedprophylactically, they can decrease the amount of hematopoietic cellsthat would be lost in the subject versus the amount lost if the subjectwere treated with the inhibitor but not with the agent. If usedprophylactically or acutely, the agents can shorten the time forrecovery of a hematopoietic cell-type to at least protective levels, andpreferably to normal levels, versus the length of time which would passbefore protective or normal levels were achieved if the subject weretreated with the inhibitor but not with the agent.

Generally, doses of active compounds of the present invention would befrom about 0.01 mg/kg per day to less than 1 mg/kg per day. A variety ofadministration routes are available. The methods of the invention,generally speaking, may be practiced using any mode of administrationthat is medically acceptable, meaning any mode that produces effectivelevels of the active compounds without causing clinically unacceptableadverse effects. Such modes of administration include oral, rectal,topical, nasal, interdermal, or parenteral routes. The term “parenteral”includes subcutaneous, intravenous, intramuscular, or infusion.Intravenous or intramuscular routes are not particularly suitable forlong-term therapy and prophylaxis. They could, however, be preferred inemergency situations. Oral administration is preferred for theconvenience to the patient as well as the dosing schedule. SeeRemington's Pharmaceutical Sciences, 18th edition, 1990, pp 1694-1712;incorporated by reference). Those of skill in the art can readilydetermine the various parameters and conditions for producing dosageswithout resort to undue experimentation.

Compositions suitable for oral administration may be presented asdiscrete units, such as capsules, tablets, lozenges, each containing apredetermined amount of the active agent. Other compositions includesuspensions in aqueous liquids or non-aqueous liquids such as a syrup,elixir or an emulsion.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like. Lower doses will result from other forms ofadministration, such as intravenous administration. In the event that aresponse in a subject is insufficient at the initial doses applied,higher doses (or effectively higher doses by a different, more localizeddelivery route) may be employed to the extent that patient tolerancepermits. Multiple doses per day are contemplated.

The agents may be combined, optionally, with apharmaceutically-acceptable carrier. The term“pharmaceutically-acceptable carrier” as used herein means one or morecompatible solid or liquid filler, diluents or encapsulating substanceswhich are suitable for administration into a human. The term “carrier”denotes an organic or inorganic ingredient, natural or synthetic, withwhich the active ingredient is combined to facilitate the application.The components of the pharmaceutical compositions also are capable ofbeing co-mingled with the molecules of the present invention, and witheach other, in a manner such that there is no interaction which wouldsubstantially impair the desired pharmaceutical efficacy.

When administered, the pharmaceutical preparations of the invention areapplied in pharmaceutically-acceptable amounts and inpharmaceutically-acceptably compositions. Such preparations mayroutinely contain salt, buffering agents, preservatives, compatiblecarriers, and optionally other therapeutic agents. When used inmedicine, the salts should be pharmaceutically acceptable, butnon-pharmaceutically acceptable salts may conveniently be used toprepare pharmaceutically-acceptable salts thereof and are not excludedfrom the scope of the invention. Such pharmacologically andpharmaceutically-acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulfuric,nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic,succinic, and the like. Also, pharmaceutically acceptable salts can beprepared as alkaline metal or alkaline earth salts, such as sodium,potassium or calcium salts.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the agent, increasing convenience to the subject andthe physician. Many types of release delivery systems are available andknown to those of ordinary skill in the art. They include polymer basesystems such as poly(lactide-glycolide), copolyoxalates,polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyricacid, and polyanhydrides. Microcapsules of the foregoing polymerscontaining drugs are described in, for example, U.S. Pat. No. 5,075,109.Delivery systems also include non-polymer systems that are: lipidsincluding sterols such as cholesterol, cholesterol esters and fattyacids or neutral fats such as mono- di- and tri-glycerides; hydrogelrelease systems; sylastic systems; peptide based systems; wax coatings;compressed tablets using conventional binders and excipients; partiallyfused implants; and the like. Specific examples include, but are notlimited to: (a) erosional systems in which the agent is contained in aform within a matrix such as those described in U.S. Pat. Nos.4,452,775, 4,667,014, 4,748,034 and 5,239,660 and (b) difusional systemsin which an active component permeates at a controlled rate from apolymer such as described in U.S. Pat. Nos. 3,832,253, and 3,854,480. Inaddition, pump-based hardware delivery systems can be used, some ofwhich are adapted for implantation.

Use of a long-term sustained release implant may be particularlysuitable for treatment of chronic conditions. Long-term release, areused herein, means that the implant is constructed and arranged todelivery therapeutic levels of the active ingredient for at least 30days, and preferably 60 days. Long-term sustained release implants arewell-known to those of ordinary skill in the art and include some of therelease systems described above.

EXAMPLES

We have demonstrated in a series of in vivo studies that the agentValboroPro (PT-100), has the ability to shorten myelosuppression causedby chemotherapy in mice. In these studies, mice were injectedintraperitoneally with a sublethal dose of 220 mg/kg cyclophosphamide(Day 1). This treatment reproducibly induced a nadir in blood cellcounts by Day 4. After 72 hours (Day 3) mice were divided into 3 groups.One group received PT-100, at the concentrations indicated, by gavage orby subcutaneous administration (s.c.), one group received G-CSF by s.c.injections and the third group received saline as a control, either byoral gavage or by s.c. injections. G-CSF was used at 0.04 ug/dose (4μg/kg/day) which is the dose frequently used in published reportsstudying the G-CSF effects in mice and is also the equivalent dose usedin cancer patients. All administrations were performed twice daily(b.i.d.) for 5 consecutive days or as indicated. Blood samples weretaken from individual mice on Day 4-8, and in some experiments on Days13 or 17. At each time pont four or five test animals were sampled.Total and differential white blood cell counts of Gimsa-stained bloodsmears were performed.

PT-100 Dose Response for Regeneration of Neutrophil

For data presented in FIG. 2, cyclophosphamide treated mice received 0.1μg, 2 μg or 5 μg/b.i.d. of PT-100 or saline by oral gavage twice dailyfor 5 consecutive days starting on Day 3 post cyclophosphamide treatmentand continuing through Day 7. In mice that received 2 or 5 μg/b.i.d.PT-100 recovery of neutrophils reproducibly preceded recovery of salinetreated mice by 1 or 2 days, while 0.1 μg/b.i.d. of PT-100 did notsignificantly enhance neutrophil recovery over saline. Normal levels ofabsolute neutrophil counts (ANC) were reached on Day 5 for micereceiving 2 μg or 5 μg/b.i.d. of PT-100, while saline treated mice didnot reach normal levels until Day 7. On Day 5 mice had received a totalof 4 doses of PT-100 (on Days 3 and 4). Additional administration ofPT-100 on Days 5, 6 and 7 caused a further increase in ANC.

The effect of PT-100 on neutrophil recovery when administered by s.c.route was very similar to that seen when administered orally. For datashown in FIG. 3 mice were injected s.c. with doses of PT-100 rangingfrom 1 to 20 μg/b.i.d. for 5 days and blood cell counts determined onDays 4 through 8, and on Day 17. For mice receiving 5 μg, 10 μg, or 20μg/b.i.d. PT-100, neutrophil recovery was accelerated over that observedin the saline treated mice. A dose of 1 μg/b.i.d. PT-100 did not showmuch effect. After termination of treatment with PT-100, In conclusion,PT-100 accelerates neutrophil regeneration in cyclophosphamide treatedmice.

Comparison of PT-100 and G-SCF Effects on Neutrophil Regeneration

G-CSF is currently used to accelerate neutrophil recovery in cancerpatients undergoing chemotherapy. The effects of G-CSF in mice are wellestablished and can be used as a reference for elucidating the mechanismby which PT-100 stimulates hematopoiesis in mice. FIG. 4 shows data froman experiment in which the effects of PT-100 and G-CSF on neutrophilregeneration are compared. Cyclophosphamide treated mice wereadministered 2 μg/b.i.d. of PT-100 by gavage or 0.04 μg/b.i.d. of G-CSF(the dose equivalent used in patients and most commonly used inpublished reports for murine studies) by subcutaneous injections for 5consecutive days starting on Day 3. Blood cell counts were performed onDays 4-8, and on Day 13.

PT-100 and G-CSF treated mice stimulated neutrophil regeneration to asimilar level during the treatment period. After treatment was stopped,ANC decreased to normal counts by Day 13. Although PT-100 has a verysimilar effect on neutrophil reconstitution, the mechanism of action isdifferent from that of G-CSF. Not only does PT-100 target a differentcellular receptor (CD 26), it also has been shown to stimulate growth ofearly human hematopoietic progenitor cells which are not affected byG-CSF.

Dose Numbering of PT-100 Administration

To determine the dose numbering of administration for optimal recoveryof neutrophils, PT-100, at indicated doses, was administered s.c. tocyclophosphamide treated mice, either once or twice per day over a fiveday period, starting on Day 3 post cyclophosphamide treatment. As shownin FIG. 5, for both doses, a twice daily administration resulted in afaster rate of neutrophil recovery to higher neutrophil levels than onceper day administration.

Duration of PT-100 Administration

In the experiments described above mice had been treated with PT-100 for5 consecutive days. To determine whether a shorter period of treatmentwith PT-100 was sufficient for the recovery of neutrophils 5 μg, 2 μg,or 1 μg/b.i.d. (six hours apart) of PT-100 was administered tocyclophosphamide treated mice by gavage for 1, 2, 3, or 5 days startingon Day 3 post cyclophosphamide treatment. Blood counts were obtained ondays 4 through 8.

Administration of PT-100 for one day was sufficient to cause anaccelerated reconstitution of neutrophils over saline treated animals.However, additional administrations of PT-100 for 2 or 3 days increasedthe rate of recovery even further. Data for the 5 μg dose are shown inFIG. 6.

Continued administration of PT-100 for a total of 4 or 5 days does notsignificantly increase the rate of neutrophil recovery or the ANC overthat achieved with 3 day administrations (data for 2 μg/b.i.d. are shownin FIG. 7).

Results shown in FIGS. 6 and 7 indicate that the PT-100 effect on theregeneration of neutrophils occurs early during treatment and continuesuntil ANC between 1000 and 1400 are achieved. Repeated administrationsaffect the kinetics of neutrophil restoration during the early periodbut does not significantly alter the ANC reached after 3 days ofadministration.

In conclusion, PT-100 accelerates neutrophil reconstitution over thatseen with saline even after a one day of treatment. An acceleratedreconstitution of neutrophils is obtained with each additional day oftreatment for up to three days. A fourth or fifth day of treatment didnot significantly increase ANC or the kinetics of reconstitution.

Human Hematopoietic Cell Responses In Vitro

Hematopoiesis is sustained by a pool of hematopoietic stem cells (HSCs)that can self-renew and differentiate into hematopoietic progenitorcells (HPCs). HPCs are committed to specific lineages which can beidentified based on their colony morphology when grown in semi-solidmedia in vitro, typically over a 2 week period. The colonies grown inthe semi-solid colony assay are functionally defined as colony- orburst-forming units and include BFU-E and CFU-E (cells committed to theerythroid lineage), CFU-GM (cells committed to thegranulocytic/monocytic lineage), BFU-MK and CFU-MK (cells committed tothe megakaryocyte lineage) and CFU-GEMM (multipotent progenitors).Although the semi-solid colony assay is a valuable tool to identifyfactors, such as G-CSF, which affect terminal differentiation, it doesnot assess the proliferative potential or self renewing properties ofthe primitive hematopoietic progenitor cells (PHPCs) (Dexter, T. A. etal., Acta Hemat. 62:299-305 (1979); Chen, B. P. et al., ImmunologicalReviews: 15 7:41-51 (1997)).

An assay to evaluate the effect of a compound or of growth factors onPHPCs was first described by Dexter (Dexter T. M. et al, J. Cell.Physiol. 91:335-344 (1977)), and combines the Long-Term Culture (LTC)with the semi-solid colony assay. LTC is initiated over a pre-formedstromal cell layer which provides the necessary hematopoietic growthfactors. It has been used extensively for the in vitro examination ofmurine and human hematopoiesis and to evaluate the ability of testcompounds to generate LTC-ICs.

The effect of PT-100 on growth of human hematopoietic cells was examinedin the 2 week CFU and the 4 and 5 week LTC assays using human bonemarrow, apheresed peripheral blood or umbilical cord blood cells. PT-100did not stimulate the generation of CFUs in the 2 week semi-solid assay,indicating that PT-100 does not affect the differentiation of committedprogenitor cells into mature blood cells. It also suggests, that themechanism and the cellular targets for PT-100 for the stimulation ofneutrophil regeneration in vivo is different from that of G-CSF whichhas been shown to stimulate CFU formation in this assay. In the LTCassays, which test for effects on early progenitor cells, PT-100significantly increased the growth of very early progenitor cells fromall three cell sources. Moreover, the data suggest that the effect ofPT-100 is on PHPCs as increases in LTC-ICs were observed at 4 weeks(FIG. 8) 5 weeks and 6 weeks (data not shown) in culture. At this timeless primitive hematopoietic progenitor cells have undergone terminaldifferentiation and lost the ability to form colonies in semi-solidcultures.

For the LTC assays, CD34⁺ cells were isolated by positive selection fromhuman bone marrow cells, apheresed peripheral blood or umbilical cordblood using a MAC separation system. To establish a stromal feederlayer, human bone marrow cells were cultured in Myelocult long termculture medium for 2 weeks. One day prior to use, the adherent stromalcells were cultured overnight with indicated concentrations of PT-100 inLTC medium and irradiated. Isolated CD34⁺ cells were overlaid onto thestromal cell layer and incubated for 30 days in the absence or presenceof indicated amounts of PT-100. Medium and PT-100 was exchanged everythree days thereafter. At the end of the culture period the culture wasassayed for progenitor cells by plating in semi-solid medium(methylcellulose) supplemented with growth factors (Stem Cell Factor,GM-CSF, IL-3 and Erythropoietin).

The total number of myeloid, erythroid, blast forming and multilineageclonogeneic progenitors (colonies CFU-GM, CFU-E, BFU-E and CFU-GEMM,respectively) were determined after 14 days in methylcellulose culture.

Data showing in FIG. 8 for a human bone marrow culture indicate thatduring a 4 week LTC assay, PT-100 increased, in a dose dependent manner,the number of clonogeneic progenitors which are able to form colonies insemi-solid medium. This suggests that PT-100 stimulates growth ofprimitive hematopoietic progenitor cells.

In similar fashion CD34⁺ cells purified from apheresed peripheral bloodor umbilical cord blood were cultured on irradiated primary stromalcells for 30 days. As had been observed with bone marrow cells, PT-100increased the number of 4 and 5 week LTC-ICs from peripheral andumbilical cord blood to very similar levels, indicating the PT-100 isable to stimulate primitive hematopoietic progenitor cell growth fromthese cell sources as well (data not shown).

PT-100 Does Not Stimulate Differentiation of Committed Progenitor Cells

Human bone marrow cells were enriched for CD34+ cells and 200 CD34+cells per well were incubated in serum free x-vivo 15 medium(Biowhittaker) with or without the indicated concentrations of PT-100for 4 hours at 37° C. The pre-incubated CD34+ cells were added to 0.9%methylcellulose in Iscove's MDM containing sub-optimal concentrations ofrecombinant human growth factors (5 ng/ml Stem Cell Factor, 1 ng/mlGM-CSF, 1 ng/ml IL-3, 0.3 units/ml Erythropoietin (Stem CellTechnologies Vancouver, BC). PT-100 was added to the medium at the sameconcentrations used for the pre-incubation. The methylcellulose mixturewas plated in duplicate in 35 mm dishes and incubated for 14 days at 37°C. Progenitor colonies (CFU-E, CFU-GM, CFU-GEMM and BFU-E) were countedunder an inverted microscope. PT-100 did not stimulate differentiationof these committed progenitor cells.

Stimulation of Hematopoiesis in the Spleen of Normal Mice

6-8 week old female BALB/c mice were administered either saline orPT-100 twice daily for 5 days at the indicated doses via eithersubcutaneous injection or oral gavage. On the sixth day the animals weresacrificed and their spleens were excised using sterile procedures. Thespleens were disrupted to produce single cell suspensions which weresubsequently treated with a solution of Tris ammonium chloride (pH 7.2)to lyse erythrocytes. The resulting splenocyte populations were in ahemocytometer and resuspended at 5×10⁶ cells/mL in Iscove's ModifiedEagles medium (IMDM) supplemented with 2% heat inactivated fetal calfserum. 0.3 mL of each splenocyte solution was added to 3 mL ofMethocult™GF M3434 (Stem Cell Technologies, Vancouver, BC, Canada), amethylcellulose medium containing recombinant cytokines used for colonyassays of murine progenitor cells. The medium was vigorously mixed andthen 1.1 mL of the mixture was placed in duplicate onto sterile 35 mmdiameter culture dishes, resulting in 5×10⁵ splenocytes/plate. Theplated cells were incubated at 37° C. under humidified conditions in 95%air/5% CO₂ for 7 days. CFU-E were enumerated as per the manufacturersspecifications after 2 days, while BFU-E, CFU-GM and CFU-GEMM wereenumerated after 7 days. For each mouse, the absolute CFU/spleen werecalculated using the total splenocyte count determined in thehemocytometer. The data shown in FIG. 9 represents the mean ±SDCFU/spleen from 3 mice in each dosing group. PT-100 stimuatedhematopoiesis for all progenitor colony types tested.

PT-100 Induces Production of G-CSF From Human Bone Marrow Stromal Cells

Mononuclear cells were purified from bone marrow and cultured long-termculture medium, (Stem Cell Technologies, Inc., Vancouver, B.C.) for 2weeks, with a single feeding of fresh medium after 1 week. Theestablished stromal cells were removed by trypsin-EDTA digest and seededinto a 35 mm tissue culture plate at 10⁶ cells per well in 1 ml ofmedium containing 10⁻⁵ M PT-100 or medium alone as control. Culturemedia were collected on day 1. Supernatants were assayed for human G-CSFusing a Quantikine high sensitivity immuno-assay kit (R+D Systems,Minneapolis, Minn.). FIG. 10 depicts the effect of PT-100 on theproduction of G-CSF by cultured human stomal cells. PT-100 stimulatesproduction of G-CSF by such cells.

The manufacture of L-VAL-R-boroPro is described in a number of publishedprocedures (Kelly, T. A., et al. J. Am. Chem. Soc. 1993.115:12537-12638; Coutts, S. J., et al., J. Med. Chem. 1996.39:2087-2094; Beak, P., et al., Tetrahedon Letters, 1989, 30:1197; Bean,F. R., et al., J. Amer. Chem. Soc. 1932. 54:4415). Pure isomers arepreferred. See also U.S. Pat. Nos. 4,935,493 and 5,462,928, thedisclosures of which are incorporated here by reference.

While the invention has been described with respect to certainembodiments, it should be appreciated that many modifications andchanges may be made by those of ordinary skill in the art withoutdeparting from the spirit of the invention. It is intended that suchmodifications, changes, and equivalents fall within the scope of thefollowing claims.

We claim:
 1. A method for treating a subject to stimulate hematopoiesisof hematopoietic cells other than mature lymphocytes in the subject,comprising: administering to a subject in need of such treatment anamount of an agent effective to increase the number of hematopoieticcells other than mature lymphocytes in the subject, wherein the agent isadministered orally in an amount less than 1 mg/kg body weight per dayand wherein the agent is a compound of Formula I:

 wherein m is an integer between 0 and 10, inclusive; A and A₁ areL-amino acid residues such that the A in each repeating bracketed unitcan be the same or a different amino acid residue; the C bonded to B isin the L-configuration; the bonds between A and N, A₁ and C, and betweenA₁ and N are peptide bonds; and each X₁ and X₂ is, independently, ahydroxyl group or a group capable of being hydrolyzed to a hydroxylgroup in aqueous solution at physiological pH.
 2. The method of claim 1,wherein the subject has an abnormally low level of hematopoietic cellsand wherein the agent is administered in an amount effective to restorelevels of a hematopoietic cell-type to a preselected normal orprotective level.
 3. The method of claim 1, wherein the subject isadministered at least two doses of the agent in an 18 hour period. 4.The method of claim 2, wherein the subject is neutropenic and whereinsaid amount is effective to restore a preselected normal or protectivelevel of neutrophils.
 5. The method of claim 2, wherein the subject hasabnormally low levels of erythrocytes and wherein said amount iseffective to restore a preselected normal or protective level oferythrocytes.
 6. The method of claim 2, wherein the subject hasabnormally low levels of platelets and wherein said amount is effectiveto restore a preselected normal or protective level of platelets.
 7. Amethod for shortening the time that a subject has an abnormally lowlevel of hematopoietic or mature blood cells resulting from treatmentwith a hematopoietic cell inhibitor, comprising: administering to asubject in need of such treatment an agent in an amount effective toincrease the number of hematopoietic cells or mature blood cells in thesubject, wherein the administration of the agent is prior to orsubstantially simultaneous with administration of the hematopoietic cellinhibitor, wherein the agent is administered orally in an amount lessthan 1 mg/kg body weight per day and wherein the agent is a compound ofFormula I:

 wherein m is an integer between 0 and 10, inclusive; A and A₁ areL-amino acid residues such that the A in each repeating bracketed unitcan be the same or a different amino acid residue; the C bonded to B isin the L-configuration; the bonds between A and N, A₁ and C, and betweenA₁ and N are peptide bonds; and each X₁ and X₂ is, independently, ahydroxyl group or a group capable of being hydrolyzed to a hydroxylgroup in aqueous solution at physiological pH.
 8. The method of claim 7,wherein the administration of the hematopoietic cell inhibitor causes anabnormally low level of hematopoietic cells in the subject and whereinthe agent is administered in an amount effective to restore levels of ahematopoietic cell-type to a preselected normal or protective level. 9.The method of claim 8, wherein the subject is administered at least twodoses of the agent in an 18 hour period.
 10. The method of claim 8,wherein the hematopoietic cell inhibitor reduces in the subjectneutrophils and wherein the amount is effective to restore in thesubject a preselected normal or protective level of neutrophils.
 11. Themethod of claim 8, wherein the hematopoietic cell inhibitor reduces inthe subject erythrocytes and wherein the amount is effective to restorein the subject a preselected normal or protective level of erythrocytes.12. A method for treating a subject to increase the number ofhematopoietic cells other than mature lymphocytes in the subjectcomprising: administering to a subject in need of such treatment anamount of an agent effective to increase hematopoietic cells other thanmature lymphocytes in the subject, wherein the agent is administered ina first regimen consisting of 2 doses or 3 doses in an 18 hour period,wherein the agent is administered orally in an amount less than 1 mg/kgbody weight per day and wherein the agent is a compound of Formula I:

 wherein m is an integer between 0 and 10, inclusive; A and A₁ areL-amino acid residues such that the A in each repeating bracketed unitcan be the same or a different amino acid residue; the C bonded to B isin the L-configuration; the bonds between A and N, A₁ and C, and betweenA₁ and N are peptide bonds; and each X₁ and X₂ is, independently, ahydroxyl group or a group capable of being hydrolyzed to a hydroxylgroup in aqueous solution at physiological pH.
 13. The method of claim1, 2, 3, 4, 5 or 6, wherein the agent is ValBoroPro.
 14. The method ofclaim 8, wherein the hematopoietic cell inhibitor reduces in the subjectplatelets and wherein the amount is effective to restore in the subjecta preselected normal or protective level of platelets.
 15. The method ofclaim 7, 8, 9, 10, 11 or 14, wherein the agent is ValBoroPro.
 16. Themethod of claim 12, wherein the agent is administered in a secondregimen consisting of 2 doses or 3 doses in an 18 hours period, andwherein, the second regimen is separate in time from the first regimen.17. The method of claim 16, wherein the agent is administered in a thirdregimen consisting of 2 doses or 3 doses in an 18 hours period, andwherein, the third regimen is separate in time from the first and secondregimens.
 18. The method of claim 17, wherein the agent is administeredin a fourth regimen consisting of 2 doses or 3 doses in an 18 hoursperiod, and wherein, the fourth regimen is separate in time from thefirst, second, and third regimens.
 19. The method of claim 18, whereinthe agent is administered in a fifth regimen consisting of 2 doses or 3doses in an 18 hours period, and wherein, the fifth regimen is separatein time from the first, second, third and fourth regimens.
 20. Themethod of claim 12, 16, 17, 18 or 19, wherein the subject has abnormallylow neutrophils and, wherein said amount is effective to restore apreselected normal or protective level of neutrophils.
 21. The method ofclaim 12, 16, 17, 18 or 19, wherein the agent is ValBoroPro.